The sarcoplasmic reticulum (SR) functions, among other things, as a specialized intracellular calcium (Ca2+) store. Channels in the SR called ryanodine receptors (RyRs) open and close to regulate the release of Ca2+ from the SR into the intracellular cytoplasm of the cell. Release of Ca2+ into the cytoplasm from the SR increases cytoplasmic Ca2+ concentration. Open probability (Po) of the RyR receptor refers to the likelihood that the RyR channel is open at any given moment, and therefore capable of releasing Ca2+ into the cytoplasm from the SR.
There are three types of ryanodine receptors, all of which are Ca2+ channels: RyR1, RyR2, and RyR3. RyR1 is found predominantly in skeletal muscle as well as other tissues, RyR2 is found predominantly in the heart as well as other tissues, and RyR3 is found in the brain as well as other tissues. The RyR channels are formed by four RyR polypeptides in association with four FK506 binding proteins (FKBPs), specifically FKBP12 (calstabin1) and FKBP12.6 (calstabin2). Calstabin1 binds to RyR1, calstabin2 binds to RyR2, and calstabin1 binds to RyR3. The FKBP proteins (calstabin1 and calstabin2) bind to the RyR channel (one molecule per RyR subunit), stabilize RyR-channel functioning, and facilitate coupled gating between neighboring RyR channels, thereby preventing abnormal activation of the channel during the channel's closed state.
Protein kinase A (PKA) binds to the cytoplasmic surface of the RyR receptors. PKA phosphorylation of the RyR receptors causes partial dissociation of calstabins from RyRs. Dissociation of calstabin from RyR causes increased open probability of RyR, and therefore increased Ca2+ release from the SR into the intracellular cytoplasm.
Ca2+ release from the SR in skeletal muscle cells and heart cells is a key physiological mechanism that controls muscle performance, because increased concentration of Ca2+ in the intracellular cytoplasm causes contraction of the muscle.
Excitation-contraction (EC) coupling in skeletal muscles involves electrical depolarization of the plasma membrane in the transverse tubule (T-tubule), which activates voltage-gated L-type Ca2+ channels (LTCCs). LTCCs trigger Ca2+ release from the SR through physical interaction with RyR1. The resulting increase in cytoplasmic Ca2+ concentration induces actin-myosin interaction and muscle contraction. To enable relaxation, intracellular Ca2+ is pumped back into the SR via SR Ca2+-ATPase pumps (SERCAs), which is regulated by phospholamban (PLB) depending on the muscle fiber type.
It has been shown that disease forms that result in sustained activation of the sympathetic nervous system and increased plasma catecholamine levels cause maladaptive activation of intracellular stress pathways resulting in destabilization of the RyR1 channel closed state and intracellular Ca2+ leak. SR Ca2+ leak via RyR1 channels was found to deplete intracellular SR calcium stores, to increase compensatory energy consumption, and to result in significant acceleration of muscle fatigue. The stress-induced muscle defect permanently reduces isolated muscle and in vivo performance particularly in situations of increased demand.
It also has been shown that destabilization of the RyR1 closed state occurs under pathologic conditions of increased sympathetic activation and involves depletion of the stabilizing calstabin1 (FKBP12) channel subunit. Proof-of-principle experiments have shown that PKA activation as an end effector of the sympathetic nervous systems increases RyR1 PKA phosphorylation at Ser-2843 which decreases the binding affinity of calstabin1 to RyR1 and increases channel open probability.
In cardiac striated muscle, RyR2 is the major Ca2+-release channel required for EC coupling and muscle contraction. During EC coupling, depolarization of the cardiac-muscle cell membrane during phase zero of the action potential activates voltage-gated Ca2+ channels. Ca2+ influx through the open voltage-gated channels in turn initiates Ca2+ release from the SR via RyR2. This process is known as Ca2+-induced Ca2+ release. The RyR2-mediated, Ca2+-induced Ca2+ release then activates the contractile proteins in the cardiac cell, resulting in cardiac muscle contraction.
Phosphorylation of cardiac RyR2 by PKA is an important part of the “fight or flight” response that increases cardiac EC coupling gain by augmenting the amount of Ca2+ released for a given trigger. This signaling pathway provides a mechanism by which activation of the sympathetic nervous system, in response to stress, results in increased cardiac output. PKA phosphorylation of RyR2 increases the open probability of the channel by dissociating calstabin2 (FKBP12.6) from the channel complex. This, in turn, increases the sensitivity of RyR2 to Ca2+-dependent activation.
Despite advances in treatment, heart failure remains an important cause of mortality in Western countries. An important hallmark of heart failure is reduced myocardial contractility. In heart failure, contractile abnormalities result, in part, from alterations in the signaling pathway that allows the cardiac action potential to trigger Ca2+ release via RyR2 channels and muscle contraction. In particular, in failing hearts, the amplitude of the whole-cell Ca2+ transient is decreased and the duration prolonged.
Cardiac arrhythmia, a common feature of heart failure, results in many of the deaths associated with the disease. Atrial fibrillation (AF) is the most common cardiac arrhythmia in humans, and represents a major cause of morbidity and mortality. Structural and electrical remodeling—including shortening of atrial refractoriness, loss of rate-related adaptation of refractoriness, and shortening of the wavelength of re-entrant wavelets—accompany sustained tachycardia. This remodeling is likely important in the development, maintenance and progression of atrial fibrillation. Studies suggest that calcium handling plays a role in electrical remodeling in atrial fibrillation.
Approximately 50% of all patients with heart disease die from fatal cardiac arrhythmias. In some cases, a ventricular arrhythmia in the heart is rapidly fatal—a phenomenon referred to as “sudden cardiac death” (SCD). Fatal ventricular arrhythmias and SCD also occur in young, otherwise-healthy individuals who are not known to have structural heart disease. In fact, ventricular arrhythmia is the most common cause of sudden death in otherwise-healthy individuals.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disorder in individuals with structurally normal hearts. It is characterized by stress-induced ventricular tachycardia—a lethal arrhythmia that causes SCD. In subjects with CPVT, physical exertion and/or stress induce bidirectional and/or polymorphic ventricular tachycardias that lead to SCD even in the absence of detectable structural heart disease. CPVT is predominantly inherited in an autosomal-dominant fashion. Individuals with CPVT have ventricular arrhythmias when subjected to exercise, but do not develop arrhythmias at rest. Studies have identified mutations in the human RyR2 gene, on chromosome 1q42-q43, in individuals with CPVT.
Failing hearts (e.g., in patients with heart failure and in animal models of heart failure) are characterized by a maladaptive response that includes chronic hyperadrenergic stimulation. In heart failure, chronic beta-adrenergic stimulation is associated with the activation of beta-adrenergic receptors in the heart, which, through coupling with G-proteins, activate adenylyl cyclase and thereby increase intracellular cAMP concentration. CAMP activates cAMP-dependent PKA, which has been shown to induce hyperphosphorylation of RyR2. Thus, chronic heart failure is a chronic hyperadrenergic state that results in several pathologic consequences, including PKA hyperphosphorylation of RyR2.
The PKA hyperphosphorylation of RyR2 has been proposed as a factor contributing to depressed contractile function and arrhythmogenesis in heart failure. Consistent with this hypothesis, PKA hyperphosphorylation of RyR2 in failing hearts has been demonstrated, in vivo, both in animal models and in patients with heart failure undergoing cardiac transplantation.
In failing hearts, the hyperphosphorylation of RyR2 by PKA induces the dissociation of FKBP12.6 (calstabin2) from the RyR2 channel. This causes marked changes in the biophysical properties of the RyR2 channel, including increased open probability (Po) due to an increased sensitivity to Ca2+-dependent activation; destabilization of the channel, resulting in subconductance states; and impaired coupled gating of the channels, resulting in defective EC coupling and cardiac dysfunction. Thus, PKA-hyperphosphorylated RyR2 is very sensitive to low-level Ca2+ stimulation, and this manifests itself as a diastolic SR Ca2+ leak through the PKA hyperphosphorylated RyR2 channel.
The maladaptive response to stress in heart failure results in depletion of FKBP12.6 from the channel macromolecular complex. This leads to a shift to the left in the sensitivity of RyR2 to Ca2+-induced Ca2+ release, resulting in channels that are more active at low-to-moderate Ca2+ concentrations. Over time, the increased “leak” through RyR2 results in resetting of the SR Ca2+ content to a lower level, which in turn reduces EC coupling gain and contributes to impaired systolic contractility.
Additionally, a subpopulation of RyR2 that are particularly “leaky” can release SR Ca2+ during the resting phase of the cardiac cycle, diastole. This results in depolarizations of the cardiomyocyte membrane known as delayed after-depolarizations (DADs), which are known to trigger fatal ventricular cardiac arrhythmias.
In patients with CPVT mutations in their RyR2 and otherwise structurally-normal hearts, a similar phenomenon is at work. Specifically, it is known that exercise and stress induce the release of catecholamines that activate beta-adrenergic receptors in the heart. Activation of the beta-adrenergic receptors leads to PKA hyperphosphorylation of RyR2 channels. Evidence also suggests that the PKA hyperphosphorylation of RyR2 resulting from beta-adrenergic-receptor activation renders mutated RyR2 channels more likely to open in the relaxation phase of the cardiac cycle, increasing the likelihood of arrhythmias.
Cardiac arrhythmias are known to be associated with diastolic SR Ca2+ leaks in patients with CPVT mutations in their RyR2 and otherwise structurally-normal hearts. In these cases, the most common mechanism for induction and maintenance of ventricular tachycardia is abnormal automaticity. One form of abnormal automaticity, known as triggered arrhythmia, is associated with aberrant release of SR Ca2+, which initiates DADs. DADs are abnormal depolarizations in cardiomyocytes that occur after repolarization of a cardiac action potential. The molecular basis for the abnormal SR Ca2+ release that results in DADs has not been fully elucidated. However, DADs are known to be blocked by ryanodine, providing evidence that RyR2 plays a key role in the pathogenesis of this aberrant Ca2+ release.
It has been shown that exercise-induced arrhythmias and sudden death (in patients with CPVT) result from a reduced affinity of FKBP12.6 (calstabin2) for RyR2. Additionally, it has been demonstrated that exercise activates RyR2 as a result of phosphorylation by adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase (PKA). Mutant RyR2 channels, which had normal function in planar lipid bilayers under basal conditions, were more sensitive to activation by PKA phosphorylation—exhibiting increased activity (open probability) and prolonged open states, as compared with wild-type channels. In addition, PKA-phosphorylated mutant RyR2 channels were resistant to inhibition by Mg2+, a physiological inhibitor of the channel, and showed reduced binding to FKBP12.6 (aka calstabin2, which stabilizes the channel in the closed state). These findings indicate that, during exercise, when the RyR2 are PKA-phosphorylated, the mutant CPVT channels are more likely to open in the relaxation phase of the cardiac cycle (diastole), increasing the likelihood of arrhythmias triggered by SR Ca2+ leak.
Fatigue is the process whereby skeletal muscles become weaker with repeated or intense use such as exercise, or as a result of an illness, disorder or disease. Fatigue can result in task failure and it can be a pronounced symptom in a variety of medical conditions including heart failure, renal failure, cancer, and various muscular dystrophies. Over the past decade, it has become evident that the two dominant classical explanations of muscle fatigue, namely, accumulation of lactic acid and intracellular acidosis, do not cause fatigue. In fact, both may be protective mechanisms during high intensity exercise to prevent fatigue (Allen and Westerblad 2004; Pedersen, Nielsen et al. 2004).
Muscle contraction depends on the efficient coupling of electrical stimulation of the muscle surface to Ca2+ release via the ryanodine receptor, the SR Ca2+ release channel, to the generation of actinmyosin cross bridges. It is evident, then, that a defect in excitation-contraction coupling that resulted in a reduction in the amplitude of the Ca2+ transient would, among other effects, result in impaired contraction and force generation through ineffective myosin cross bridge formation. Eberstein and Sandow suggested inhibition of Ca2+ release as a likely factor in the fatigue process (Eberstein and Sandow 1963). Reductions in the amplitude of Ca2+ release evoked during fatiguing stimulation have been reported in multiple muscle preparations (Allen, Lee et al. 1989; Westerblad and Allen 1991; Allen and Westerblad 2001). The time course of recovery from fatigue parallels the time course over which prolonged depression of Ca2+ release is observed (Westerblad, Bruton et al. 2000).
SR Ca2+ leak was documented in myofibers following intense exercise and in a model of muscular dystrophy (Wang, Weisleder et al. 2005), possibly due to defective skeletal ryanodine receptors (RyR1s). Chronic activation of the sympathetic nervous system (SNS) in the context of heart failure promotes intrinsic skeletal muscle fatigue due to depletion of the phosphodiesterease PDE4D3 from the RyR1 complex, RyR1 PKA hyperphosphorylation at Serine 2844, calstabin1 depletion from the RyR1 complex, and a gain-of-function channel defect (Reiken, Lacampagne et al. 2003). RyR1 dysfunction in the skeletal muscle leads to altered local subcellular Ca2+ release events and impaired global calcium transients (Ward, Reiken et al. 2003). JTV-519, (4-[3-(4-benzylpiperidin-1-yl)propionyl]-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine monohydrochloride-a 1,4-benzothiazepine has been shown to be a modulator of RyR calcium-ion channels), given in the context of a murine model of heart failure, was able to improve skeletal muscle function, as assessed by an ex vivo isolated muscle fatiguing protocol, five weeks after left coronary artery ligation. JTV-519's beneficial effect on muscle fatigue was not solely due to cardiac improvement, as a beneficial effect was still seen when the drug was given to calstabin2 deficient mice which derive no cardiac benefit from treatment with JTV-519. Thus, it has been postulated that JTV-519 directly affects muscle function (Wehrens, Lehnart et al. 2005). In the context of chronic exercise, identical changes in the RyR1 macromolecular complex, namely depletion of PDE4D3 from the RyR1 complex, RyR1 PKA hyperphosphorylation at Serine 2844, and calstabin1 depletion from the RyR1 complex are related in a time-dependent and activity-dependent manner with repeated intense exercise in a mouse model. These biochemical changes in the RyR1 macromolecular complex regulation and function are stable following prolonged exercise and recover slowly over days to weeks. It has therefore been proposed that RyR1 Ca2+ leak limits peak muscle performance and mediates muscle damage during prolonged, stressful exercise.
The contraction of striated muscle is initiated when calcium (Ca2+) is released from tubules within the muscle cell known as the sarcoplasmic reticulum (SR). Calcium release channels, called ryanodine receptors (RyR), on the SR are required for excitation-contraction (EC) coupling. The type 2 ryanodine receptor (RyR2) is found in the heart, while the type 1 ryanodine receptor (RyR1) is found in skeletal muscle. The RyR1 receptor is a tetramer comprised of four 565,000 dalton RyR1 polypeptides and four 12,000 dalton FK-506 binding proteins (FKBP12). FKBP12s are regulatory subunits that stabilize RyR channel function (Brillantes et al., 1994) and facilitate coupled gating between neighboring RyR channels (Marx et al., 1998); the latter are packed into dense arrays in specialized SR regions that release intracellular stores of Ca2+, thereby triggering muscle contraction. In addition to FKBP12, the RyR1 macromolecular complex also includes the catalytic and regulatory subunits of PKA, and the phosphatase PP1 (Marx et al., 2001).
One FKBP12 molecule is bound to each RyR1 subunit. Dissociation of FKBP12 significantly alters the biophysical properties of the channels, resulting in the appearance of subconductance states, and increased open probability (Po) of the channels (Brillantes et al., 1994; Gaburjakova et al., 2001). In addition, dissociation of FKBP12 from RyR1 channels inhibits coupled gating resulting in channels that gate stochastically rather than as an ensemble (Marx et al., 1998). Coupled gating of arrays of RyR channels is thought to be important for efficient EC coupling that regulates muscle contraction (Marx et al., 1998). FKBPs are cis-trans peptidyl-prolyl isomerases that are widely expressed and subserve a variety of cellular functions (Marks, 1996). FKBP12s are tightly bound to and regulate the function of the skeletal (RyR1) (Brillantes et al., 1994; Jayaraman et al., 1992) and cardiac (RyR2) (Kaftan et al., 1996) muscle Ca2+ release channels, as well as a related intracellular Ca2+ release channel known as the type 1 inositol 1,4,5-triphosphate receptor (IP3R1) (Cameron et al., 1997), and the type I transforming growth factor β (TGFβ) receptor (TβRI) (Chen et al., 1997).
U.S. Pat. No. 7,312,044, the contents of which are hereby incorporated by reference, discloses methods of treating defective skeletal muscle function during heart failure by administering an agent which inhibits dissociation of FKB12 binding protein from RyR1 receptor.
Co-pending U.S. patent application Ser. No. 11/212,309 and U.S. Pat. No. 7,704,990, the contents of which are hereby incorporated by reference, disclose methods of making and using novel benzothiazepine derivatives to treat and prevent disorders and diseases associated with the RyR receptors, including skeletal muscular disorders and diseases such as skeletal muscle fatigue, exercise-induced skeletal muscle fatigue, muscular dystrophy, bladder disorders, and incontinence.
There is a need to identify new agents effective for treating or preventing muscle fatigue that is stress or exercise induced or that results from diseases associated with the RyR receptors that regulate calcium channel functioning in cells, including cardiac disease or disorder, defective skeletal muscle function, HIV Infection, AIDS, muscular dystrophy, cancer, malnutrition, exercise-induced muscle fatigue, age-associated muscle fatigue, renal disease, and renal failure.